This proposal requests additional funding for the last two-year period of Jun Wu's K01 and to facilitate her transition into an independent investigator in metabolic research. The profound health consequences associated with obesity emphasize the importance of developing effective therapeutic interventions. Obesity is essentially a disorder of energy balance, in which intake exceeds expenditure. Broadly, adipocytes have been divided into white and brown fat cells. White fat cells are specialized to store chemical energy, whereas brown fat defends against hypothermia and obesity through thermogenesis mediated by mitochondrial uncoupling protein 1 (UCP1). The funded K award has led to the isolation of beige cells from murine white fat depots. Different from the classical brown fat, whic is derived from a myf-5+ muscle-like cellular lineage, beige fat cells, dispersing in white fat tissues are from a non-myf-5 lineage. Beige cells have a gene expression pattern distinct from either white or brown fat. Beige cells resemble white fat cells in having extremely low basal expression of Ucp1, but like classical brown fat, they respond to cyclic AMP stimulation with high Ucp1 expression and respiration rates. Primary beige adipocytes are preferentially sensitive to the polypeptide hormone irisin, which induces browning of the white adipose depots and improves glucose homeostasis. Deposits of UCP-1+ adipocytes previously observed in adult humans molecularly resemble murine beige adipose cells much more closely than brown. The overall goal of this proposal is to study molecular regulation of beige fat through beige-selective markers and hormonal responses; and how beige fat impacts systematic metabolic homeostasis in humans. Aim #1. Previous studies shown that activation of beige marker CD137 will increase energy expenditure in rodents, but the mechanism is unknown. How CD137 activation/inactivation affects beige fat function will be investigated in vitro and in vivo. Aim #2. Molecules known to induce browning of the white fat depots will be investigated for the molecular mechanisms how they affect beige adipocytes function. Beige-selective hormones/signaling molecules will be identified and functionally characterized. Gene set enrichment analysis will be conducted to illustrate downstream signaling cascades of beige selective hormonal response(s). Aim #3. Preliminary data shown a robust upregulation of thermogenic program upon irisin treatment in the differentiated cultures of primary stromal vascular fraction (SVF) from various human adipose depots, suggesting that beige precursors/adipocytes may exist in these adipose depots in humans. Primary beige precursors will be isolated from both supraclavicular region and subcutaneous adipose tissues (liposuction samples) using the established beige cell sorting protocols and human beige fat functions will be investigated.